A simple ex vivo bioassay for 5-FU transport into healthy buccal mucosal cells

Fluorouracil (5-FU), a chemotherapeutic agent widely used in the treatment of numerous common malignancies, causes oral mucositis in a proportion of patients. The contribution of drug transport processes to the development of this toxicity is currently unknown. This work aimed to establish and optimise a simple phenotyping assay for 5-FU uptake into primary buccal mucosal cells (BMC). The uptake kinetics of radiolabelled 5-FU were determined in pooled BMC freshly collected from healthy volunteers. The inter- and intra-individual variability in 5-FU uptake was then assessed across a cohort that included both healthy volunteers and cancer patients. 5-FU uptake into pooled primary BMC was both time and concentration dependent. An Eadie–Hofstee analysis suggested two components; a high-affinity (KM = 3.3 µM) low-capacity ( $$V_{\text{MAX}}$$ = 57.8 pmol min−1 105 viable cells−1) transporter, and a high-capacity ( $$V_{\text{MAX}}$$ = 1230 pmol min−1 105 viable cells−1) low-affinity (KM = 3932 µM) transporter. There was 180-fold variation in the rate of 5-FU uptake into BMC (0.10–17.86 pmol min−1 105 viable cells−1) across the 34 subjects (healthy participants N = 24, cancer patients N = 10). Notably, retesting of a subset of these participants (N = 16) multiple times over a period of up to 140 days demonstrated poor stability of the uptake phenotype within individuals. The uptake of 5-FU into healthy oral mucosal cells is a highly variable process facilitated by membrane transporters at pharmacologically relevant concentrations. This bioassay is simple, minimally invasive, and suitable for phenotypic analysis of drug transport in healthy primary cells.